Despite the importance of smooth muscle in health and disease, many details of its function and how it is regulated remain unclear. This renewal application is a continued effort to gain more insights into this area. The overall goal is to use molecular and cellular methodologies to obtain information on the functional roles of various smooth muscle proteins and to enhance our understanding of the regulatory mechanism of smooth muscle contraction. The program contains five projects, one facility core and one administrative core. Projects IA and IB deal with the thick filament regulation by investigating the mechanisms of myosin light chain kinase and myosin light chain phosphatase, respectively, by which the level of myosin activation is regulated. Project II studies the structure and function of caldesmon to elucidate its roles in the thin filament-based regulation of smooth muscle contraction. Project III is a new initiative to gain high-resolution structural information on the regulatory proteins in smooth muscles, starting with actin-caldesmon and myosin light chain phosphatase. In Project IV the integrated system of the thick and the thin filaments will be examined, and the roleof tropomyosin in the cooperative properties of smooth muscle thin filament will be explored. This highly interactive and multi-disciplinary program utilizes a wide spectrum of different approaches, including analytical ultracentrifugation,chemical crosslinking, fluorescence resonance energy transfer, circular dichroism, isothermal titration calorimetry, site-directed mutagenesis, X-ray crystallography, physiological measurements of smooth muscle tissues, and gene knockout experiments. In addition, rapid kinetics, chemical and enzymatic proteolysis, synthetic peptides, mass spectrometry and immuno-electron microscopy will also be used. In particular, Project II proposes to use segmental isotope labeling techniques to study the structure of caldesmon by NMR spectroscopy. All subprojects will be supported by a Biophysical/Biochemical Core, which provides protein chemistry, analytical ultracentrifugation and electron microscopy and immunocytochemistry services. Funding of this program will enable us to expand our current investigation on the regulatory mechanisms in the smooth muscle system and lead to possible therapeutic measures of smooth muscle related diseases.